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Correlation of Quantitative Ultrasound of Bone with Biochemical Markers of Bone Resorption in Women with Osteoporotic Fractures
Journal of Clinical Densitometry, vol. 2, no. 3, 231–239, Fall 1999 © Copyright 1999 by Humana Press Inc. All rights of any nature whatsoever reserved. 0169-4194/99/2:231–239/$12.25
Original Article
Correlation of Quantitative Ultrasound of Bone with Biochemical Markers of Bone Resorption in Women with Osteoporotic Fractures Carmelo E. Fiore, MD, PHD, Pietra Pennisi, MD, Maurizio Gibilaro, MD, Salvatore Di Fazzio, MD, Daniela Impellizzieri, MD, and Maria Grazia Ramirez, RTR Clinica Medica II, University School of Medicine, University of Catania, Italy
Abstract The purpose of this study was to examine the correlation of parameters of bone quality assessed by quantitative ultrasound (QUS) with biochemical indexes of bone resorption. QUS of the calcaneus and the hand phalanges, and biochemical parameters (urinary excretion of pyridinoline [Pyr] and deoxypyridinoline [D-Pyr]) were measured in a group of 30 well-characterized postmenopausal women with established osteoporosis and fragility fractures. All patients were treatment free. QUS data significantly correlated with both urinary Pyr and D-Pyr (p < 0.001 for speed of sound [SOS], broadband ultrasound attenuation [BUA], and stiffness at the heel; p < 0.001 for amplitude-dependent SOS at the proximal phalanges of the nondominant hand). No significant correlation was observed between spine and femoral bone mineral density and the urinary excretion of Pyr and D-Pyr. Results of this study suggest that QUS of bone evaluates characteristics of bone influenced by the bone resorption rate. Key Words: Bone mineral density; quantitative ultrasound; bone resorption rate; established osteoporosis.
investigators has been generally directed toward densitometric measurements (bone mineral content, bone mineral density [BMD]) obtained on both axial and appendicular sites by means of single photon absorptiometry (SPA), single X-ray absorptiometry, dual X-ray absorptiometry (DXA), and quantitative computed tomography (2–5). All these techniques provide data significantly correlated with fracture risk, even though a large overlap of bone density values between women with and without fractures has been reported by several investigators (6,7). The recent observation that bone strength depends on both bone mass and architecture, as well as bone elasticity (8),
Introduction Osteoporosis is currently defined as a syndrome characterized by a reduction of bone mass and a deterioration of skeletal microarchitecture leading to an increase of bone fragility and possibility of fractures (1). Although the reduction of bone mass is only one of the numerous factors that may influence bone fragility, the interest of the majority of
Received 12/22/98; Revised 03/01/99; Accepted 03/22/99. Address correspondence to Prof. Carmelo E. Fiore, Clinica Medica II OVE, University of Catania, Via Plebiscito 628— 95124 Catania, Italy. E-mail: [email protected]
231
232 supported the development of noninvasive measurement techniques that give information on other bone fragility factors distinct from bone mass. Among these, quantitative ultrasound (QUS) measurement techniques have been recognized as useful tools for noninvasive assessment of skeletal status. Recent evidence demonstrates that ultrasound parameters such as speed of sound (SOS) and broadband ultrasound attenuation (BUA) provide information on both quantitative and qualitative aspects of bone tissue (9–11), because they reflect also nonmass properties of bone and detect a fragility component distinct from bone mass (12). For these reasons, it is not surprising that the clinical observation that correlation between BMD measurements and ultrasound parameters is generally weak, although each of these two techniques shows an independent ability to discriminate patients with hip fractures and vertebral deformities attributable to osteoporosis (13,14). Recent experimental evidence suggests that QUS parameters are associated not only with bone density but also connectivity parameters and anisotropy (orientational dependence of connectivity basically dominated by the mineral spread in a collagen framework) (15–19). However, according to other investigators (20), organic matrix would contribute very little to BUA in vitro, which would be influenced mainly by the presence of bone mineral. The recent observation by Kawana et al. (21) about the existence of a positive correlation among ultrasound parameters (SOS, stiffness, and BUA) and some biochemical indicators of bone turnover (osteocalcin and urinary D-Pyr) in the early postmenopausal period suggests that US measurements can be influenced by the bone turnover rate evaluated by means of sensitive indicators. Since this observation would attribute to ultrasound measurements a discriminating ability greater than the more traditional techniques, we deemed it of interest to compare the power of the correlation between ultrasound and BMD measures and plasmatic and urinary concentrations of some factors indicating the degree of bone turnover in a group of postmenopausal women with established osteoporosis and fragility fractures. Journal of Clinical Densitometry
Fiore et al.
Materials and Methods Thirty postmenopausal caucasian women aged 62–88 yr (mean 75.3 ± 6.39) were selected among patients who attended the Laboratory for the Metabolic Bone Disease at the Vittorio Emanuele Hospital in Catania. The study included women who gave written informed consent and received preliminary approval by the local ethical committee. Exclusion criteria were nonosteoporotic fractures after severe trauma and current or past therapy with any medication known to affect bone metabolism, including calcium supplements. All women were living at home. A group of 30 age-matched healthy women was used as a control. Lateral thoracic and lumbar spine radiographs were obtained on each patient using a standardized procedure; the diagnosis of vertebral fracture was made by quantitatively assessing the vertebral body, according to Genant et al. (22).
BMD Measurements Density at the lumbar spine (L1–L4) was determined by DXA using the Hologic QDR 1000 (Hologic, Walthman, MA). Two measurements were made with the patient repositioned between measurements. The coefficient of variation (CV) for the lumbar spine was 0.68. The BMD of the femur was measured by the same apparatus. The neck and the Ward’s triangle regions were measured. The CVs were 1.82 for the neck BMD and 2.4% for the Ward’s triangle BMD.
Ultrasound Measurements Ultrasound measurements were performed at the heel and the fingers. We used the Sahara Clinical Bone Sonometer device (Hologic) to measure the ultrasonic SOS and the BUA of the calcaneus. These two measured values are calculated and combined by the Sahara system into a third parameter, the quantitative ultrasound index, termed stiffness. This index, different from the biomechanical term defined by biomechanicists, is considered a sensitive measure of bone status because it combines the sensitivity of both BUA and SOS, while simultaneously reducing precision error. The values of stiffness normally range from 0 to 150, with higher values obtained for young healthy subjects and lower values obtained for older osteoporotic patients. The Volume 2, 1999
Ultrasound and Bone Resorption short-term precision estimated by the CV was 0.22% for SOS, 1.18% for BUA, and 1.34% for stiffness. Two measurements of the same side were obtained on all subjects; duplicate scans appear to be crucial because they enable us to reduce the errors by performing a third scan if the measurements differ by 10 dB/MHz for BUA, and 5 ms for SOS. In the two patients with hip fracture, the measurements were performed on both heels. Amplitude-dependent ultrasound velocity (ADSOS) through the proximal phalanges of the fingers was measured by means of a DBM Sonic 1200 (Igea, Carpi, Italy). This investigation assesses the velocity at which ultrasound crosses the distal metaphyses of the first phalanxes of the last four fingers (23). The value of the AD-SOS was reported as the mean value of the four measurements. In vivo short-term precision was 0.77% (24).
Measurements of Biochemical Markers Blood samples, obtained on the morning after an overnight fast, and 24-h preservative-free urine specimens were stored at –20°C in multiple aliquots. Concentration of serum and urinary calcium, and phosphorus and urinary creatinine, were measured using standard laboratory methods. Bone-specific alkaline phosphatase (BAP) was measured in serum by an enzyme-linked immunosorbent assay (ELISA) Alkphase-B™ (Metra Biosystem, Mountain View, CA), which uses a monoclonal antibody (MAb) with selective, high affinity for the bone AP isoform and low crossreactivity (5%) to the liver form of AP (25). The binding of intestinal and placental isoenzymes is negligible. The sensitivity of the assay is 0.7 U/L; the intra- and interassay coefficients of variation are less than 10%. The normal reference range of BAP for postmenopausal women is 15–43 U/L. Urinary-free Pyr was measured by a competitive enzyme immunoassay (Pyrilinks, Metra Biosystem) based on MAb that demonstrates specificity for the free form of Pyr crosslinks (26). Pyr results are corrected for urinary concentration by creatinine (Cr) and are expressed as nmol of Pyr/mmol of Cr. The intra- and interassay CVs are 7 and 11.2%, respectively. Normal values in healthy postmenopausal women range from 16.0 to 37.0 nmol/mmol. Urinary-free D-Pyr was performed with an ELISA Journal of Clinical Densitometry
233 (Pyrilinks-D, Metra Biosystem) that utilizes a monoclonal anti-D-Pyr antibody with <2.5% crossreactivity with free Pyr (27). The intra- and interassay CVs are <10%. Values obtained for D-Pyr are corrected by urinary Cr and are expressed as nmol of DPyr/mmol of Cr. Reference values in healthy postmenopausal women range from 3.0 to 7.4 nmol/mmol. The results of the biochemical assays are expressed as the mean ± 1 SD. The various correlations were assessed by linear regression analysis and comparison between groups by nonparametric tests.
Results Table 1 presents the clinical characteristics for the subjects in this study. One or two vertebral fractures were present in 28 women (93%), and femoral neck fracture was present in two women (6.6%). The average age of women was 75.3 yr, and the mean age at menopause was nearly 50 yr. Body weight and Body Mass Index were within the range of normal values. The mean BMD at each site measured (spine, femoral neck, and Ward’s triangle) was significantly lower than in control subjects. As for ultrasound measurements, all parameters were significantly lower in patients than in controls subjects (SOS, BUA, and stiffness at calcaneus, and SOS at phalanx; p < 0.001). Biochemical indicators of bone resorption were significantly altered (as a mean) in all patients when compared to values from control subjects. The calcium/Cr urinary ratio, and the concentration of urinary-free Pyr and free D-Pyr were significantly higher than those measured in control subjects (p < 0.001). Urinary Cr excretion was not significantly different in osteoporotic women and control subjects; this minimizes the possibility that Cr-corrected values of urinary bone resorption markers measured in osteoporotic women may appear misleadingly higher than in control subjects as a consequence of lower urinary Cr values. Moreover, correlations between urinary Cr and Pyr and D-Pyr was not significant (r2 = 0.1693; p = 0.1838 and r2 = 0.07626; p = 0.3899, respectively). Correlation coefficients between ultrasound and DXA parameters were calculated in all patients. Correlation was also calculated between Pyr/Cr, DVolume 2, 1999
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Table 1 Mean Values (±SD) of the Metabolic, Ultrasonic, and Densitometric Parameters, and Demographic Data of the 30 Postmenopausal Osteoporotic Women with Fragility Fracturesa Osteoporotic women with fragility fractures (n = 30) Age (yr) Age at menopause (yr) Height (cm) Weight (kg) BMI (kg/m2) UCr (mmol/L) UCa/Cr BAP (U/L) UPyr/Cr (nmol/mmol) UD-Pyr/Cr (nmol/mmol) Ultrasound parameters AD-SOS phalanx (m/s) SOS calcaneus (m/s) Stiffness index (calcaneus) BUA calcaneus (db/MHz) BMD (mg/cm2) Spine Femoral neck Ward’s triangle
Age-matched healthy controls (n = 30)
75.33 ± 6.39 49.93 ± 4.97 147.2 ± 6.23 59.96 ± 9.92 21.4 ± 6 5.40 ± 2.92 0.98 ± 0.42b 27.64 ± 7.6 51.47 ± 18.79b 10.17 ± 3.12b
73.44 ± 6.68 48.45 ± 4.66 150 ± 6.6 57.88 ± 10.76 22.1 ± 5.4 5.19 ± 2.67 0.34 ± 0.28 29.7 ± 8.2 31.6 ± 9.8 5.6 ± 1.8
1755 ± 95.41c 1507 ± 17.40c 66.70 ± 12.21c 45.03 ± 10.95c
2024 ± 58.9 1537 ± 33 82.8 ± 14 98 ± 12
729 ± 106.5c 572 ± 78.67c 391 ± 78.93c
936 ± 139 763 ± 128 654 ± 112
a Results are compared to the mean values of 30 age-matched healthy controls. BMI, Body Mass Index; UCr, urinary creatinine; UCa/Cr, urinary calcium/creatinine ratio; BAP, bone-specific alkaline phosphatase; UPyr/Cr, urinary pyridinoline/creatinine ratio; UDPyr/Cr, (urinary deoxypyridinoline/creatinine ratio; AD-SOS amplitude-dependent speed of sound; SOS, speed of sound; BUA, broadband ultrasound attenuation; BMD, bone mineral density. b Significantly higher (p < 0.001) than age-matched healthy controls (Wilcoxon test). c Significantly lower (p < 0.001) than age-matched healthy controls (Wilcoxon test).
Pyr/Cr ratios, and ultrasound and DXA measurements. Correlations between ultrasound and DXA parameters were not significant. No correlation was seen between both spine and femoral BMD and the urinary excretion of Pyr and D-Pyr (Fig. 1). Figures 2 and 3 show the results of the correlation studies of SOS, BUA, and stiffness vs the urinary excretion of pyridinoline and deoxypyridinoline. The observed correlation coefficients were all significantly different from 0 (from 0.26 to 0.74). There was a tendency toward higher correlation comparing indexes of bone resorption to the SOS measured at the phalangeal site, whereas the correlation between the same parameters at the calcaneus appeared to be less strong. Serum values of BAP were within the normal range in all patients; since this did not preclude corJournal of Clinical Densitometry
relations with BMD or ultrasound, r2 and p values were calculated for spine and femoral BMD and ultrasound parameters. Results indicated that BAP serum concentrations were not significantly correlated with bone mass in our osteoporotic patients (r2 < 0.013; p > 0.20 for all parameters).
Discussion In this cross-sectional analysis of postmenopausal women with established osteoporosis and fragility fractures, we found, as expected, that both ultrasound and DXA measurements were significantly lower than in the control group. Although the numbers reported in this study are small, the results might be of interest with respect to the ability of the ultrasound approach to reflect changes in the elasticVolume 2, 1999
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Fig. 1. Plot of spine, femoral neck, and femoral Ward’s triangle BMD against urinary excretion of free pyridinoline (Pyr/Cr) and free deoxypyridinoline (D-Pyr/Cr). No significant correlation was observed (linear regression).
ity of bone that depends on changes of the bone organic matrix. Our small cohort of well-characterized postmenopausal women (all were treatment free, and all had one or two osteoporotic fractures) seems to be a suitable model to evaluate the interrelationships among the rate of bone turnover, bone loss, and bone material properties. We found no strong correlation of ultrasonic parameters with DXA measurements at both cortical and trabecular sites. This is consistent with the very weak correlaJournal of Clinical Densitometry
tion reported by Schott et al. (13) in a group of 43 osteoporotic women with a hip fracture, and with the data by Reid et al. (28); but it is in contrast with results of some other published reports (29–31). This disagreement could be owing to the fact that most positive studies evaluated correlation in healthy subjects, with normal bone density and structure, whereas studies in osteoporotic patients were not able to show a clear association between the two measures. Volume 2, 1999
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Fig. 2. Linear relationship between the Stiffness Index and BUA of the calcaneus, and the urinary excretion of free pyridinoline (Pyr/Cr) and free deoxypyridinoline (D-Pyr/Cr).
In fact, there is a growing body of evidence, that ultrasound measures more of bone integrity than only bone mass, showing that simple correlation with BMD is not the appropriate method of comparison (12,32–34). Since there is general agreement that ultrasound discriminates patients with fractures as well as DXA measurements and that ultrasound velocity is as good as SPA in estimating odds of fracture (35), it would be of interest to understand whether ultrasound measurements can discriminate patients with the highest bone turnover, who are likely to lose more bone than those with low bone resorption markers, and who are therefore at risk for further fragility fractures. Some discrepancies in the literature about the rate of bone turnover in the elderly are likely to be owing to the use of nonbone-specific markers (especially as far as bone resorption is concerned) and ill-defined criteria of inclusion in the studies. We measured the
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urinary excretion of Pyr and D-Pyr, which are recognized to be two of the most specific and sensitive markers of bone resorption, and generally well correlated with bone resorption measured by bone histomorphometry (36–38). The efficacy of crosslink-based markers of bone collagen degradation in detecting small changes in bone metabolism (39) makes it reasonable to consider them as a gold standard against which other measurements should be compared. The lack of a significant correlation between metabolic and densitometric data obtained by DXA measurements in our patients would seem to be in contrast with the association between high bone turnover and decreased bone mass reported by Ravn et al. (40). These investigators observed that in postmenopausal women, the difference in bone mass between the highest and the lowest quartiles of the urinary Cross-Laps (an assay that measures type I
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Fig. 3. Linear relationship between the SOS of the phalanx and the calcaneus, and the urinary excretion of free pyridinoline (Pyr/Cr) and free deoxypyridinoline (D-Pyr/Cr).
collagen degradation products in urine) was 8–14%. This discrepancy could be explained by the fact that we calculated correlation among individual values of BMD and biochemical parameters; whereas patients had in fact a very low BMD value as a group (corresponding to high mean values of Pyr and D-Pyr excretion), the individual numbers did not correlate, in the sense that the patients with the lowest BMD of the whole group did not have (as one might be tempted to guess) the highest Pyr and D-Pyr urinary excretion. We concluded that BMD measurements were not influenced by the bone turnover rate in osteoporotic patients, but were able to just reflect the presence of bone mineral and bone quantity. On the contrary, if we compare individual ultrasound measures with individual Pyr and D-Pyr urinary excretion, the correlation appears to be highly significant, thus demonstrating a strong
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association between ultrasound velocity and the bone turnover rate in osteoporotic patients. These results confirm the ability of ultrasound measures to discriminate between normal and osteoporotic bone, and suggest that changes in the organic matrix, as subtle as those evaluated by changes in bone collagen degradation products in the serum (38,39), may influence properties of bone such as elasticity and strength. The main limitations of this study are the relatively small number of subjects and the crosssectional nature of the analysis. The latter does not allow us to rule out whether some of the fracture cases have had recent fractures, and that the biochemical markers might be elevated as a result of localized fracture healing, because we do not know whether the same results would have been observed prior to fracture. Nevertheless we believe that even if the markers are elevated as a result of fracture, the
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238 correlation with ultrasound could indicate that the entire skeleton is compromised, rather than the local fracture site. These results are in agreement with data obtained in vitro by Mehta et al. (41), which show that a progressive breakdown of the organic matrix in cortical bone specimens results in substantial changes in the elasticity of bone measured by ultrasound. If we consider that elasticity is a determinant of the resistance of bone to external loads, we may speculate that measuring ultrasound velocity and biochemical markers of bone resorption may complement BMD measures and could lead to a more detailed evaluation of bone status and possibly the risk of further fractures in patients with established osteoporosis.
Acknowledgment
Fiore et al.
11.
12. 13.
14.
15.
16.
17.
This work was supported by the Italian Ministry of the University and Scientific Research.
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Ultrasound and Bone Resorption 27. Robbins SP, Woitge H, Hessley R, Ju J, Seyedin SM, Seibel MJ. 1994 Direct enzyme-limited immunoassay for urinary deoxypyridinoline as a specific marker for measuring bone resorption. J Bone Miner Res 9:1643–1649. 28. Reid DM, Massie A, Porter RV. 1991 Bone mineral density and structure: comparison of hip and spine dual energy x-ray absorptiometry with broadband ultrasound attenuation for osteoporosis screening. Osteoporos Int 1:194–198. 29. Agren M, Karellas A, Leahey D, Marks S, Baran D. 1991 Ultrasound attenuation of the calcaneus: a sensitive and specific discriminator of osteopenia in postmenopausal women. Calcif Tissue Int 48:240–244. 30. Baran DT, McCarthy CK, Leahey D, Lew R. 1991 Broadband ultrasonic attenuation of the calcaneus predicts lumbar and femoral neck density in caucasian women: a preliminary study. Osteoporos Int 1:110–113. 31. Lees B, Stevenson JC. 1993 Preliminary evaluation of a new ultrasound bone densitometer. Calcif Tissue Int 53:149–152. 32. Glüer CC, Vahlensiec M, Foulkner KG, Engelke K, Black D, Genant HK, 1992 Site-matched calcaneal measurements of broad-band ultrasound attenuation and single x-ray absorptiometry: do they measure different skeletal properties? J Bone Miner Res 9:1071–1079. 33. Turner CH, Peacock M, Timmermann L, Neal JM, Johnston CC Jr. 1995 Calcaneal ultrasonic measurements discriminate hip fracture independently of bone mass. Osteoporos Int 5:130–135.
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239 34. Bauer DC, Gluer CC, Genant HK, Stone K. 1995 Quantitative ultrasound and vertebral fracture in postmenopausal women. J Bone Miner Res 10:353–368. 35. Stegman MR, Heaney RP, Recker RR. 1995 Comparison of speed of sound ultrasound with single photon absorptiometry for determining fracture odds ratios. J Bone Miner Res 10:346–352. 36. Garnero P, Sornay-Rendu E, Chapuy MC, Delmas PD. 1996 Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res 11:337–349. 37. Delmas PD, Schlemmer A, Gineyts E, Riis B, Christiansen C. 1991 Urinary excretion of pyridinoline crosslinks correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis. J Bone Miner Res 6:639–644. 38. Knott L, Bayley AJ. 1998 Collagen cross-links in mineralizing tissues: a review of their chemistry, function and clinical relevance. Bone 22:181–187. 39. Russel RGG. 1997 The assessment of bone metabolism in vivo usimg biochemical approach. Horm Metab Res 29:138–144. 40. Ravn P, Fledelius C, Rosenquist C, Overgaard K, Christiansen C. 1996 High bone turnover is associated with low bone mass in both pre- and postmenopausal women. Bone 19:291–298. 41. Mehta SS, Öz OK, Antich PP. 1998 Bone elasticity and ultrasound velocity are affected by subtle changes in the organic matrix. J Bone Miner Res 13:114–121.
Volume 2, 1999
Original Article
Correlation of Quantitative Ultrasound of Bone with Biochemical Markers of Bone Resorption in Women with Osteoporotic Fractures Carmelo E. Fiore, MD, PHD, Pietra Pennisi, MD, Maurizio Gibilaro, MD, Salvatore Di Fazzio, MD, Daniela Impellizzieri, MD, and Maria Grazia Ramirez, RTR Clinica Medica II, University School of Medicine, University of Catania, Italy
Abstract The purpose of this study was to examine the correlation of parameters of bone quality assessed by quantitative ultrasound (QUS) with biochemical indexes of bone resorption. QUS of the calcaneus and the hand phalanges, and biochemical parameters (urinary excretion of pyridinoline [Pyr] and deoxypyridinoline [D-Pyr]) were measured in a group of 30 well-characterized postmenopausal women with established osteoporosis and fragility fractures. All patients were treatment free. QUS data significantly correlated with both urinary Pyr and D-Pyr (p < 0.001 for speed of sound [SOS], broadband ultrasound attenuation [BUA], and stiffness at the heel; p < 0.001 for amplitude-dependent SOS at the proximal phalanges of the nondominant hand). No significant correlation was observed between spine and femoral bone mineral density and the urinary excretion of Pyr and D-Pyr. Results of this study suggest that QUS of bone evaluates characteristics of bone influenced by the bone resorption rate. Key Words: Bone mineral density; quantitative ultrasound; bone resorption rate; established osteoporosis.
investigators has been generally directed toward densitometric measurements (bone mineral content, bone mineral density [BMD]) obtained on both axial and appendicular sites by means of single photon absorptiometry (SPA), single X-ray absorptiometry, dual X-ray absorptiometry (DXA), and quantitative computed tomography (2–5). All these techniques provide data significantly correlated with fracture risk, even though a large overlap of bone density values between women with and without fractures has been reported by several investigators (6,7). The recent observation that bone strength depends on both bone mass and architecture, as well as bone elasticity (8),
Introduction Osteoporosis is currently defined as a syndrome characterized by a reduction of bone mass and a deterioration of skeletal microarchitecture leading to an increase of bone fragility and possibility of fractures (1). Although the reduction of bone mass is only one of the numerous factors that may influence bone fragility, the interest of the majority of
Received 12/22/98; Revised 03/01/99; Accepted 03/22/99. Address correspondence to Prof. Carmelo E. Fiore, Clinica Medica II OVE, University of Catania, Via Plebiscito 628— 95124 Catania, Italy. E-mail: [email protected]
231
232 supported the development of noninvasive measurement techniques that give information on other bone fragility factors distinct from bone mass. Among these, quantitative ultrasound (QUS) measurement techniques have been recognized as useful tools for noninvasive assessment of skeletal status. Recent evidence demonstrates that ultrasound parameters such as speed of sound (SOS) and broadband ultrasound attenuation (BUA) provide information on both quantitative and qualitative aspects of bone tissue (9–11), because they reflect also nonmass properties of bone and detect a fragility component distinct from bone mass (12). For these reasons, it is not surprising that the clinical observation that correlation between BMD measurements and ultrasound parameters is generally weak, although each of these two techniques shows an independent ability to discriminate patients with hip fractures and vertebral deformities attributable to osteoporosis (13,14). Recent experimental evidence suggests that QUS parameters are associated not only with bone density but also connectivity parameters and anisotropy (orientational dependence of connectivity basically dominated by the mineral spread in a collagen framework) (15–19). However, according to other investigators (20), organic matrix would contribute very little to BUA in vitro, which would be influenced mainly by the presence of bone mineral. The recent observation by Kawana et al. (21) about the existence of a positive correlation among ultrasound parameters (SOS, stiffness, and BUA) and some biochemical indicators of bone turnover (osteocalcin and urinary D-Pyr) in the early postmenopausal period suggests that US measurements can be influenced by the bone turnover rate evaluated by means of sensitive indicators. Since this observation would attribute to ultrasound measurements a discriminating ability greater than the more traditional techniques, we deemed it of interest to compare the power of the correlation between ultrasound and BMD measures and plasmatic and urinary concentrations of some factors indicating the degree of bone turnover in a group of postmenopausal women with established osteoporosis and fragility fractures. Journal of Clinical Densitometry
Fiore et al.
Materials and Methods Thirty postmenopausal caucasian women aged 62–88 yr (mean 75.3 ± 6.39) were selected among patients who attended the Laboratory for the Metabolic Bone Disease at the Vittorio Emanuele Hospital in Catania. The study included women who gave written informed consent and received preliminary approval by the local ethical committee. Exclusion criteria were nonosteoporotic fractures after severe trauma and current or past therapy with any medication known to affect bone metabolism, including calcium supplements. All women were living at home. A group of 30 age-matched healthy women was used as a control. Lateral thoracic and lumbar spine radiographs were obtained on each patient using a standardized procedure; the diagnosis of vertebral fracture was made by quantitatively assessing the vertebral body, according to Genant et al. (22).
BMD Measurements Density at the lumbar spine (L1–L4) was determined by DXA using the Hologic QDR 1000 (Hologic, Walthman, MA). Two measurements were made with the patient repositioned between measurements. The coefficient of variation (CV) for the lumbar spine was 0.68. The BMD of the femur was measured by the same apparatus. The neck and the Ward’s triangle regions were measured. The CVs were 1.82 for the neck BMD and 2.4% for the Ward’s triangle BMD.
Ultrasound Measurements Ultrasound measurements were performed at the heel and the fingers. We used the Sahara Clinical Bone Sonometer device (Hologic) to measure the ultrasonic SOS and the BUA of the calcaneus. These two measured values are calculated and combined by the Sahara system into a third parameter, the quantitative ultrasound index, termed stiffness. This index, different from the biomechanical term defined by biomechanicists, is considered a sensitive measure of bone status because it combines the sensitivity of both BUA and SOS, while simultaneously reducing precision error. The values of stiffness normally range from 0 to 150, with higher values obtained for young healthy subjects and lower values obtained for older osteoporotic patients. The Volume 2, 1999
Ultrasound and Bone Resorption short-term precision estimated by the CV was 0.22% for SOS, 1.18% for BUA, and 1.34% for stiffness. Two measurements of the same side were obtained on all subjects; duplicate scans appear to be crucial because they enable us to reduce the errors by performing a third scan if the measurements differ by 10 dB/MHz for BUA, and 5 ms for SOS. In the two patients with hip fracture, the measurements were performed on both heels. Amplitude-dependent ultrasound velocity (ADSOS) through the proximal phalanges of the fingers was measured by means of a DBM Sonic 1200 (Igea, Carpi, Italy). This investigation assesses the velocity at which ultrasound crosses the distal metaphyses of the first phalanxes of the last four fingers (23). The value of the AD-SOS was reported as the mean value of the four measurements. In vivo short-term precision was 0.77% (24).
Measurements of Biochemical Markers Blood samples, obtained on the morning after an overnight fast, and 24-h preservative-free urine specimens were stored at –20°C in multiple aliquots. Concentration of serum and urinary calcium, and phosphorus and urinary creatinine, were measured using standard laboratory methods. Bone-specific alkaline phosphatase (BAP) was measured in serum by an enzyme-linked immunosorbent assay (ELISA) Alkphase-B™ (Metra Biosystem, Mountain View, CA), which uses a monoclonal antibody (MAb) with selective, high affinity for the bone AP isoform and low crossreactivity (5%) to the liver form of AP (25). The binding of intestinal and placental isoenzymes is negligible. The sensitivity of the assay is 0.7 U/L; the intra- and interassay coefficients of variation are less than 10%. The normal reference range of BAP for postmenopausal women is 15–43 U/L. Urinary-free Pyr was measured by a competitive enzyme immunoassay (Pyrilinks, Metra Biosystem) based on MAb that demonstrates specificity for the free form of Pyr crosslinks (26). Pyr results are corrected for urinary concentration by creatinine (Cr) and are expressed as nmol of Pyr/mmol of Cr. The intra- and interassay CVs are 7 and 11.2%, respectively. Normal values in healthy postmenopausal women range from 16.0 to 37.0 nmol/mmol. Urinary-free D-Pyr was performed with an ELISA Journal of Clinical Densitometry
233 (Pyrilinks-D, Metra Biosystem) that utilizes a monoclonal anti-D-Pyr antibody with <2.5% crossreactivity with free Pyr (27). The intra- and interassay CVs are <10%. Values obtained for D-Pyr are corrected by urinary Cr and are expressed as nmol of DPyr/mmol of Cr. Reference values in healthy postmenopausal women range from 3.0 to 7.4 nmol/mmol. The results of the biochemical assays are expressed as the mean ± 1 SD. The various correlations were assessed by linear regression analysis and comparison between groups by nonparametric tests.
Results Table 1 presents the clinical characteristics for the subjects in this study. One or two vertebral fractures were present in 28 women (93%), and femoral neck fracture was present in two women (6.6%). The average age of women was 75.3 yr, and the mean age at menopause was nearly 50 yr. Body weight and Body Mass Index were within the range of normal values. The mean BMD at each site measured (spine, femoral neck, and Ward’s triangle) was significantly lower than in control subjects. As for ultrasound measurements, all parameters were significantly lower in patients than in controls subjects (SOS, BUA, and stiffness at calcaneus, and SOS at phalanx; p < 0.001). Biochemical indicators of bone resorption were significantly altered (as a mean) in all patients when compared to values from control subjects. The calcium/Cr urinary ratio, and the concentration of urinary-free Pyr and free D-Pyr were significantly higher than those measured in control subjects (p < 0.001). Urinary Cr excretion was not significantly different in osteoporotic women and control subjects; this minimizes the possibility that Cr-corrected values of urinary bone resorption markers measured in osteoporotic women may appear misleadingly higher than in control subjects as a consequence of lower urinary Cr values. Moreover, correlations between urinary Cr and Pyr and D-Pyr was not significant (r2 = 0.1693; p = 0.1838 and r2 = 0.07626; p = 0.3899, respectively). Correlation coefficients between ultrasound and DXA parameters were calculated in all patients. Correlation was also calculated between Pyr/Cr, DVolume 2, 1999
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Table 1 Mean Values (±SD) of the Metabolic, Ultrasonic, and Densitometric Parameters, and Demographic Data of the 30 Postmenopausal Osteoporotic Women with Fragility Fracturesa Osteoporotic women with fragility fractures (n = 30) Age (yr) Age at menopause (yr) Height (cm) Weight (kg) BMI (kg/m2) UCr (mmol/L) UCa/Cr BAP (U/L) UPyr/Cr (nmol/mmol) UD-Pyr/Cr (nmol/mmol) Ultrasound parameters AD-SOS phalanx (m/s) SOS calcaneus (m/s) Stiffness index (calcaneus) BUA calcaneus (db/MHz) BMD (mg/cm2) Spine Femoral neck Ward’s triangle
Age-matched healthy controls (n = 30)
75.33 ± 6.39 49.93 ± 4.97 147.2 ± 6.23 59.96 ± 9.92 21.4 ± 6 5.40 ± 2.92 0.98 ± 0.42b 27.64 ± 7.6 51.47 ± 18.79b 10.17 ± 3.12b
73.44 ± 6.68 48.45 ± 4.66 150 ± 6.6 57.88 ± 10.76 22.1 ± 5.4 5.19 ± 2.67 0.34 ± 0.28 29.7 ± 8.2 31.6 ± 9.8 5.6 ± 1.8
1755 ± 95.41c 1507 ± 17.40c 66.70 ± 12.21c 45.03 ± 10.95c
2024 ± 58.9 1537 ± 33 82.8 ± 14 98 ± 12
729 ± 106.5c 572 ± 78.67c 391 ± 78.93c
936 ± 139 763 ± 128 654 ± 112
a Results are compared to the mean values of 30 age-matched healthy controls. BMI, Body Mass Index; UCr, urinary creatinine; UCa/Cr, urinary calcium/creatinine ratio; BAP, bone-specific alkaline phosphatase; UPyr/Cr, urinary pyridinoline/creatinine ratio; UDPyr/Cr, (urinary deoxypyridinoline/creatinine ratio; AD-SOS amplitude-dependent speed of sound; SOS, speed of sound; BUA, broadband ultrasound attenuation; BMD, bone mineral density. b Significantly higher (p < 0.001) than age-matched healthy controls (Wilcoxon test). c Significantly lower (p < 0.001) than age-matched healthy controls (Wilcoxon test).
Pyr/Cr ratios, and ultrasound and DXA measurements. Correlations between ultrasound and DXA parameters were not significant. No correlation was seen between both spine and femoral BMD and the urinary excretion of Pyr and D-Pyr (Fig. 1). Figures 2 and 3 show the results of the correlation studies of SOS, BUA, and stiffness vs the urinary excretion of pyridinoline and deoxypyridinoline. The observed correlation coefficients were all significantly different from 0 (from 0.26 to 0.74). There was a tendency toward higher correlation comparing indexes of bone resorption to the SOS measured at the phalangeal site, whereas the correlation between the same parameters at the calcaneus appeared to be less strong. Serum values of BAP were within the normal range in all patients; since this did not preclude corJournal of Clinical Densitometry
relations with BMD or ultrasound, r2 and p values were calculated for spine and femoral BMD and ultrasound parameters. Results indicated that BAP serum concentrations were not significantly correlated with bone mass in our osteoporotic patients (r2 < 0.013; p > 0.20 for all parameters).
Discussion In this cross-sectional analysis of postmenopausal women with established osteoporosis and fragility fractures, we found, as expected, that both ultrasound and DXA measurements were significantly lower than in the control group. Although the numbers reported in this study are small, the results might be of interest with respect to the ability of the ultrasound approach to reflect changes in the elasticVolume 2, 1999
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Fig. 1. Plot of spine, femoral neck, and femoral Ward’s triangle BMD against urinary excretion of free pyridinoline (Pyr/Cr) and free deoxypyridinoline (D-Pyr/Cr). No significant correlation was observed (linear regression).
ity of bone that depends on changes of the bone organic matrix. Our small cohort of well-characterized postmenopausal women (all were treatment free, and all had one or two osteoporotic fractures) seems to be a suitable model to evaluate the interrelationships among the rate of bone turnover, bone loss, and bone material properties. We found no strong correlation of ultrasonic parameters with DXA measurements at both cortical and trabecular sites. This is consistent with the very weak correlaJournal of Clinical Densitometry
tion reported by Schott et al. (13) in a group of 43 osteoporotic women with a hip fracture, and with the data by Reid et al. (28); but it is in contrast with results of some other published reports (29–31). This disagreement could be owing to the fact that most positive studies evaluated correlation in healthy subjects, with normal bone density and structure, whereas studies in osteoporotic patients were not able to show a clear association between the two measures. Volume 2, 1999
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Fig. 2. Linear relationship between the Stiffness Index and BUA of the calcaneus, and the urinary excretion of free pyridinoline (Pyr/Cr) and free deoxypyridinoline (D-Pyr/Cr).
In fact, there is a growing body of evidence, that ultrasound measures more of bone integrity than only bone mass, showing that simple correlation with BMD is not the appropriate method of comparison (12,32–34). Since there is general agreement that ultrasound discriminates patients with fractures as well as DXA measurements and that ultrasound velocity is as good as SPA in estimating odds of fracture (35), it would be of interest to understand whether ultrasound measurements can discriminate patients with the highest bone turnover, who are likely to lose more bone than those with low bone resorption markers, and who are therefore at risk for further fragility fractures. Some discrepancies in the literature about the rate of bone turnover in the elderly are likely to be owing to the use of nonbone-specific markers (especially as far as bone resorption is concerned) and ill-defined criteria of inclusion in the studies. We measured the
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urinary excretion of Pyr and D-Pyr, which are recognized to be two of the most specific and sensitive markers of bone resorption, and generally well correlated with bone resorption measured by bone histomorphometry (36–38). The efficacy of crosslink-based markers of bone collagen degradation in detecting small changes in bone metabolism (39) makes it reasonable to consider them as a gold standard against which other measurements should be compared. The lack of a significant correlation between metabolic and densitometric data obtained by DXA measurements in our patients would seem to be in contrast with the association between high bone turnover and decreased bone mass reported by Ravn et al. (40). These investigators observed that in postmenopausal women, the difference in bone mass between the highest and the lowest quartiles of the urinary Cross-Laps (an assay that measures type I
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Fig. 3. Linear relationship between the SOS of the phalanx and the calcaneus, and the urinary excretion of free pyridinoline (Pyr/Cr) and free deoxypyridinoline (D-Pyr/Cr).
collagen degradation products in urine) was 8–14%. This discrepancy could be explained by the fact that we calculated correlation among individual values of BMD and biochemical parameters; whereas patients had in fact a very low BMD value as a group (corresponding to high mean values of Pyr and D-Pyr excretion), the individual numbers did not correlate, in the sense that the patients with the lowest BMD of the whole group did not have (as one might be tempted to guess) the highest Pyr and D-Pyr urinary excretion. We concluded that BMD measurements were not influenced by the bone turnover rate in osteoporotic patients, but were able to just reflect the presence of bone mineral and bone quantity. On the contrary, if we compare individual ultrasound measures with individual Pyr and D-Pyr urinary excretion, the correlation appears to be highly significant, thus demonstrating a strong
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association between ultrasound velocity and the bone turnover rate in osteoporotic patients. These results confirm the ability of ultrasound measures to discriminate between normal and osteoporotic bone, and suggest that changes in the organic matrix, as subtle as those evaluated by changes in bone collagen degradation products in the serum (38,39), may influence properties of bone such as elasticity and strength. The main limitations of this study are the relatively small number of subjects and the crosssectional nature of the analysis. The latter does not allow us to rule out whether some of the fracture cases have had recent fractures, and that the biochemical markers might be elevated as a result of localized fracture healing, because we do not know whether the same results would have been observed prior to fracture. Nevertheless we believe that even if the markers are elevated as a result of fracture, the
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238 correlation with ultrasound could indicate that the entire skeleton is compromised, rather than the local fracture site. These results are in agreement with data obtained in vitro by Mehta et al. (41), which show that a progressive breakdown of the organic matrix in cortical bone specimens results in substantial changes in the elasticity of bone measured by ultrasound. If we consider that elasticity is a determinant of the resistance of bone to external loads, we may speculate that measuring ultrasound velocity and biochemical markers of bone resorption may complement BMD measures and could lead to a more detailed evaluation of bone status and possibly the risk of further fractures in patients with established osteoporosis.
Acknowledgment
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11.
12. 13.
14.
15.
16.
17.
This work was supported by the Italian Ministry of the University and Scientific Research.
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